The present invention relates to novel culture methods for the ex vivo expansion of TcRxcex3xcex4+ T cells.
TcRxcex3xcex4+ cells are a small subset of circulating T lymphocytes that are distinct from conventional TcRxcex1xcex2+ T cells which recognize, with fine specificity, foreign peptide antigens in the context of classical class I or class II major histocompatibility complex (MHC) restriction elements. By contrast, TcRxcex3xcex4+ T cells are able to recognize both peptide and non-peptide antigens which may be derived from either foreign microorganisms or endogenous cellular products induced by stress such as viral infection or transformation. Moreover, unlike antigen recognition by TcRxcex1xcex2+ T cells, antigen recognition by TcRxcex3xcex4+ T cells is not MHC-restricted.
The T cell receptors of TcRxcex1xcex2+ and TcRxcex3xcex4+ T cells are distinguished by the different genetic elements that encode them. The majority of TcRxcex3xcex4+ T cells are classified into two main subsets, Vxcex41+ and Vxcex42+, based on the genes that encode their xcex4 chain. The major subset of TcRxcex3xcex4+ T cells in human peripheral blood expresses Vxcex42 in combination with Vxcex39, while most of the remainder express Vxcex41 in combination with Vxcex32, Vxcex33, Vxcex34, Vxcex35 or Vxcex38 (Salerno, A. and Dieli, F., 1998).
Since TcRxcex3xcex4+ T cells lack the fine specificity characteristics of TcRxcex1xcex2+ T cells, it has been proposed that they represent a more primitive immune mechanism that provides a first-line surveillance function against infection and tumours (Boismenu, R. et al., 1997). Several studies have documented the response of TcRxcex3xcex4+ T cells to various viruses, bacteria and parasites (Bukowski, J. F. et al., 1994; Wallace, M. et al., 1995; Lang, F. et al., 1995; Elloso, M. M. et al., 1996) as well as their ability to mediate lysis of tumour cells of various origins (Zocchi, M. R. et al., 1990; Kitayama, J. et al., 1993; Choudhary, A. et al., 1995). Hematopoietic tumours may be particularly susceptible to the lytic effects of TcRxcex3xcex4+ T cells, since transgenic mice expressing the Vxcex31.1 transgene display spontaneous resistance to injected T cell leukemias, and TcRxcex3xcex4+ T cell hybridomas derived from these mice preferentially respond to hematopoietic malignant cells over non-hematopoietic tumour cells (Penninger, J. et al., 1995). Moreover, human TcRxcex3xcex4+ T cells clones derived from patient peripheral blood and bone marrow have been shown to lyse autologous leukemic cells in acute lymphoblastic leukemia and acute myeloid leukemia, respectively (Bensussan, A. et al., 1989; Jahn, B. et al., 1995). Furthermore, improved disease-free survival in leukemia patients after allogeneic bone marrow transplantation has been shown to be associated with an increase in the number and percentage of TcRxcex3xcex4+ T cells in peripheral blood (Lamb, L. S. et al., 1996). Collectively, these results suggest that TcRxcex3xcex4+ T cells may have therapeutic potential in the treatment of cancer and infectious diseases.
Many of the published methods describing the ex vivo expansion of TcRxcex3xcex4+ T cells require the presence of antigen. Virus-infected or transformed cells or cell lines, bacteria and parasites have been shown to stimulate TcRxcex3xcex4+ T cell expansion ex vivo, as have established tumour cell lines. For example, herpes simplex virus (HSV)-infected cells were used to stimulate the expansion of Vxcex42+ cells (Bukowski, J. F. et al., 1994), while Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines were used to stimulate the expansion of Vxcex41+ cells (Orsini, D. L. M. et al., 1993). Extracts of Mycobacterium tuberculosis and blood-stage Plasmodium falciparum malarial antigens have been shown to stimulate proliferation of TcRxcex3xcex4+ T cells (Constant, P. et al., 1994; Elloso, M. M. et al., 1996). Daudi, an immortalized human Burkitt""s lymphoma cell line, can also stimulate the proliferation of TcRxcex3xcex4+ T cells (Kaur, I. et al., 1993). In addition, well-characterized, non-peptidyl antigens of the prenyl phosphate family, for example, isopentenyl pyrophosphate, have been shown to stimulate the ex vivo expansion of TcRxcex3xcex4+ T cells (Garcia, V. E. et al., 1998). In some of these systems, the antigen-stimulated cultures of TcRxcex3xcex4+ T cells were supplemented with IL-2, IL-4 or other cytokines.
TcRxcex3xcex4+ T cells have also been expanded ex vivo from populations of tumour infiltrating lymphocytes (TIL) by culture with IL-2 (Zocchi, M. R. et al., 1990) or IL-2 in combination with immobilized anti-CD3 antibody (Kitayama, J. et al., 1993) or anti-TcRxcex3xcex4 antibody (Yu, S. et al., 1999). In these systems, selective stimulation of the TcRxcex3xcex430  T cells by the tumour antigens is presumed to have occurred in vivo prior to isolation of T cells from the cancerous tissue.
In another system, TcRxcex3xcex4+ T cells were expanded from the peripheral blood of glioblastoma patients using a solid-phase, immobilized anti-CD3 antibody in combination with IL-2 followed by culture in IL-2 alone (Yamaguchi, T., et al, 1997). These authors reported that the subsequently purified TcRxcex3xcex4+ T cells did not proliferate for more than one week in the presence of IL-2 alone and therefore, they concluded, that this method would be applicable only to short term studies. They further showed that the method resulted in the expansion and enrichment of both TcRxcex3xcex4+ and TcRxcex1xcex2+ T cells, achieving TcRxcex3xcex4+ T cell purities on the order of 28%. In a subsequent report, the same authors demonstrated that this method selectively expanded the Vxcex42+ subset (Suzuki, Y., et al, 1999).
Thus, there are limitations to cell proliferation and/or requirements for antigen stimulation in the existing methods for ex vivo culture and expansion of TcRxcex3xcex4+ T cells. Furthermore, while many papers report the expansion of the Vxcex42+ cell subset, few papers report the expansion of the Vxcex41+ cell subset and none report the simultaneous expansion of both the Vxcex42+ and Vxcex41+ T cell subsets in a single culture.
In view of the foregoing, there is a need in the art for a method to selectively culture large amounts of TcRxcex3xcex4+ cells in vitro.
The present invention provides novel methods for expanding TcRxcex3xcex4+ T cells in culture in the absence of exogenous antigen. Accordingly, the present invention provides a method for expanding TcRxcex3xcex4+ T cells in a starting sample comprising:
(1) culturing cells in the starting sample in a first culture medium comprising a T cell mitogen and at least two cytokines; and
(2) culturing the cells obtained in step (1) in a second culture medium comprising at least two cytokines to expand TcRxcex3xcex4+ T cells.
In one embodiment, the present invention provides a method for expanding TcRxcex3xcex4+ T cells in a starting sample comprising:
(1) culturing cells in the starting sample in a first culture medium comprising (a) a T cell mitogen, (b) interleukin-2 and (c) interleukin-4; and
(2) culturing the cells obtained in step (1) in a second culture medium comprising (i) interleukin-2 and (ii) interleukin-4 to expand TcRxcex3xcex4+ T cells.
In another embodiment, the present invention provides a method for expanding TcRxcex3xcex4+ T cells in a starting sample comprising:
(1) obtaining low density mononuclear cells (LDMNC) from the starting sample;
(2) culturing the cells obtained in step (1) in a first culture medium comprising (a) a T cell mitogen, (b) interleukin-2 and (c) interleukin-4; and
(3) culturing the cells obtained in step (2) in a second culture medium comprising (i) interleukin-2 and (ii) interleukin-4 to expand TcRxcex3xcex4+ T cells.
In a preferred embodiment, prior to culturing the cells in the first culture medium, the cells are depleted of non-CD4+ cells or non-TcRxcex3xcex4+ cells.
In a further embodiment, the present invention provides a method for expanding TcRxcex3xcex4+ T cells in a starting sample comprising:
(1) culturing cells in the starting sample in a first culture medium comprising a leukocyte conditioned medium; and
(2) culturing the cells obtained in step (1) in a second culture medium comprising (i) interleukin-2 and (ii) interleukin-4 to expand TcRxcex3xcex4+ T cells.
In yet another embodiment, the present invention provides a method for expanding TcRxcex3xcex4+ T cells in a starting sample comprising:
(1) obtaining low density mononuclear cells (LDMNC) from the starting sample;
(2) culturing the cells obtained in step (1) in a first culture medium comprising a leukocyte conditioned medium; and
(3) culturing the cells obtained in step (2) in a second culture medium comprising (i) interleukin-2 and (ii) interleukin-4 to expand TcRxcex3xcex4+ T cells.
The TcRxcex3xcex4+ T cells obtained by the method of the invention can be used in a variety of experimental, therapeutic and commercial applications.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.